The interaction of HIV-1 Nef with p21-activated kinase 2 (Pak2) has been proposed to play an important role in T cell activation and disease progression during viral infection. However, the mechanism by which Nef activates Pak2 and whether Pak2 or another protein in the Nef-Pak2 complex is important for Nef-mediated effects on cell signaling/activation are poorly understood. Preliminary studies identified primary HIV Nef clones that exhibited a similar capacity to downregulate CD4 and MHC-I but variable ability to associate with activated Pak2. By mutagenesis, we demonstrated that Nef amino acids at positions 85, 89, 187, 188 and 191 are critical for Pak2 association. Mutation of these Nef residues reduced or abolished association with endogenous Pak2 activity but did not affect CD4 and MHC-I downregulation. Mutation of 5C Nef residue 89 specifically abolished association with FLAG-tagged Pak2-K278R, a kinase-dead Pak2 mutant. Furthermore, compensatory covariation occurs at positions 89 and 191 when both amino acids are substituted. Since residues 85, 89, 187, 188 and 191 cluster on the surface of the Nef core domain in a region distinct from the dimerization and SH3-binding domains, we propose that these Nef residues form part of a unique binding surface specifically involved in association with Pak2. This hydrophobic binding surface may participate in an as-yet-unidentified protein-protein interaction to facilitate Pak2 activation. The overall goal of this application is to understand the mechanism by which Nef enhances T cell activation via association with Pak2 or another key cellular factor in the Nef-Pak2 complex. The specific aims are: 1) Determine whether Nef association with Pak2 is important for HIV replication in resting T cells; 2) Identify cellular proteins associated with the HIV Nef- Pak2 complex and determine which protein in the complex interacts directly with Nef; 3) Investigate the role of Pak2 association in Nef-mediated enhancement of T cell activation and whether association of Nef with Pak2- PIX complexes enhances T cell activation through regulation of Cbl. Using primary Nefs and mutants defective for Pak2 association, we will investigate the biological importance of Nef association with Pak2 for viral replication in resting T cells, characterize Nef-Pak2 complexes, identify the cellular protein that interacts directly with Nef, and investigate the functional role of proteins in this complex in Nef-mediated enhancement of T cell activation and their relationship to c-Cbl, a ubiquitin ligase that negatively regulates T cell signaling. These studies will provide important insights into functions of Nef and its interactions with cellular proteins, and have significant implications for HIV transmission and pathogenesis and for identifying new therapeutic targets. The goal of this project is to understand how the Nef protein of HIV enhances activation of T cells through its association with a host cell protein called Pak2 (p21-associated kinase 2), or another key host cell factor in the Nef-Pak2 protein complex. The studies will provide important insights into viral and host mechanisms that influence HIV transmission from person-to-person and spread within the body of infected individuals, and may also help to identify new therapeutic targets for prevention and treatment of HIV infection. [unreadable] [unreadable] [unreadable] [unreadable]